How fine root turnover functions during mangrove root zone expansion and affects belowground carbon processes

Fine roots (diameter < 2 mm) are the component of belowground biomass, which are help to maintain sediment volume and resist soil compaction in mangroves. In addition, fine root turnover contributes to belowground carbon stocks. This study focused on root zone dynamics and aimed to quantify the composition of live and dead fine roots and analyze their functions during root zone expansion and belowground carbon accumulation. Shallow surface elevation tables for measuring root zone expansion were set up in Dongzhaigang Bay of Hainan Province, China; root cores and in-growth bags for measuring fine root biomass and turnover rates were used in four typical mangrove forests. Fine root biomass contributed over 60% to belowground roots, and was mainly composed of up to 69.25% dead fine roots. Fine root turnover rates ranged from 0.10 to 0.22 per year within the four forests, showing the fastest in Bruguiera forest, followed by Kandelia forest, Sonneratia plantation, and Rhizophora forest. Root zone expansion rates ranged from 0.55 to 1.28 mm yr −1, and were positively related to live fine root biomass within the upper 50 cm layer of sediment in the four forest types (R2 = 0.625, P = 0.0022). Live fine root biomass took up less than 30.75% of belowground biomass, but remarkably supported 62.50% of root zone expansion in mangroves. Turnover rates of fine roots significantly contributed to the highly dynamic changes in the carbon processes of sub-surface sediment.


Introduction
Mangroves serve as vital forest ecosystems along tropical and subtropical coastlines that provide socioeconomic and environmental services as well as critical ecological functions (Bosire et al. 2008). As one of several oceanic "blue carbon" ecosystems, mangroves can assimilate large quantities of carbon (C) by photosynthesis and capture C in sediment (Donato et al. 2011;Mcleod et al. 2011). Roots including aerial roots and belowground roots in mangroves influence both surface and sub-surface sediment C accumulation with feedback between functional processes (Krauss et al. 2003;Spenceley 1977). Mangrove ecosystems hold large amounts of organic C in sediment; this is mainly affected by tree roots along with organic and inorganic sedimentation (Alongi 2014;Ola et al. 2018). Approximately, one-third of the total C stock in sediment was composed of dead roots; undoubtedly, dead roots were a significant C source in sediment (Ouyang et al. 2017). Fine roots in mangroves contributed ~ 90% to the annual accumulation of plant debris and affected the C stock in sediment; the abundance of fine roots decreases with sediment depth Xiong et al. 2017).
Belowground roots can be divided into live and dead roots, the proportions of which depend on the mangrove species involved, sediment depth, and environmental factors (Arnaud et al. 2021). Krauss et al. (2017) found that live root growth in sediment results in the expansion of the root zone and contributes to an increase in surface elevation. Meanwhile, Ouyang et al. (2017) found that sediment C gain showed dynamic changes based on the turnover process of fine roots. Fine root turnover in mangroves plays a more vital role than aboveground litter production in C accumulation in sediment because the latter is always affected by frequent tidal inundation . Hence, the dynamic processes of roots are an important part of revealing the patterns of C gain of mangrove ecosystems in sub-surface sediment, especially with respect to climate change worldwide. However, very few studies have addressed the expansion of the root zone in mangroves worldwide, although the C gain of roots in mangroves is important.
Fine roots play a crucial role because of their short life expectancy and rapid turnover rate ranging from 0.73 yr −1 to 1.17 yr −1 (Kalyn and Van Rees 2006). Turnover of roots is the process by which short-lived fine roots are frequently produced, die fairly rapidly, and transfer C to the sediments . Increasing fine root biomass and production will promote an accumulation of organic C in sediments (He et al. 2018;Xiong et al. 2017). Therefore, fine root systems are vital for mangrove C storage and deserve more of our attention.
Previous studies on fine roots of mangroves have mainly focused on the distribution of biomass, root decomposition, and factors that influence the distribution of biomass, such as tides, species, and nutrition (Ahmed et al. 2020;Cormier et al. 2015;Medina-Calderón et al. 2021;Xiong et al. 2017;Zhang et al. 2021). In a previous study in mature Vietnamese mangroves, minirhizotrons were used to investigate fine root production and the variation in root depth along a chronosequence; the researchers found that fine root production occurred deeper than 30 cm in sediment and decreased with forest age (Arnaud et al. 2021). Fine root production was the result of dynamic change in fine root biomass (Deng et al. 2014). Furthermore, fine root production was found to be related to sediment C and fine root turnover rates were negatively relative to the soil salinity of mangroves . The drivers of root decomposition contributed to the C budgets and indicated that C contents from dead roots were a key source of C inputs in the sediment of mangroves (Ouyang et al. 2017). Studying fine root biomass benefits further understanding their functions during root zone expansion and belowground C accumulation.
Although the fine root production and turnover in mangroves were found to be influenced by various factors, e.g., forest type, forest age, hydrology, climatic conditions, and sediment depth, little is known about the expansion of the root zone induced by fine root growth and their co-effect on the gain in sediment C. Only a few studies have at least partially focused on the root zone dynamics, fine root production, and fine root turnover in the surface layer of sediment in mangroves. Usually, the surface sediment (< 30 cm) tends to have a higher C content than deeper layers (30-100 cm), due to litterfall input, aerial root growth, and sediment accretion. These ecological processes may affect surface sediment C accumulation, and the C pool is highly variable and susceptible to environmental factors (Kathiresan 2003;Krauss et al. 2014). Thus, focusing on the fine root turnover dynamics can resolve the mechanisms of sediment C accumulation. Two key questions related to root zone dynamics in mangroves were addressed in this study: 1) How does the biomass of fine roots related to the expansion of the root zone in the sediment? 2) How much does fine root turnover contribute to the C stock in surface sediment? To answer these questions, we set up permanent plots in four typical mangrove forests in Dongzhaigang Bay of Hainan Province, China, to investigate the distribution of fine roots, and monitor the production and biomass of fine roots with in-growth bags and root cores, respectively. Surface elevation tables (SETs) have been widely used to monitor the surface elevation change on the coasts of the world . We used a shallow surface elevation table (shallow-SET) to monitor the root zone expansion in these mangroves. We aimed to reveal the conditions in the expansion of the root zone following the growth of fine roots, and quantify the C gain of sub-surface sediments from fine root turnover in mangrove forests.

Study site
The study site was located at the Dongzhaigang Mangrove Nature Reserve (19°51′24″N, 110°36′49″E) in Haikou of Hainan Province, China (Figs. 1 and 2). Dongzhaigang Bay is characterized by a subtropical maritime monsoon climate with an annual mean rainfall of approximately 1676 mm and an annual temperature of approximately 24.8 °C. The tidal is irregular semidiurnal with an average tidal range of 1.6-1.8 m (Chen et al. 2009). The salinity of seawater or sediment at the study sites was approximately 10-15 PSU (practical salinity units). Kandelia obovata, Rhisphora stylosa, Bruguiera sexangula, Aegiceras corniculatum, Avicennia marina, and Ceriops tagal are the main mangrove forest species in Dongzhaigang Bay Reserve. Sonneratia apetala was introduced to China and planted in Dongzhaigang Bay in 1985. The morphological difference among mangrove aerial root structures-prop roots, pneumatophores, knee roots, plank roots. We selected four mangrove forest types of S. apetala (with pneumatophores), K. obovata (with plank roots), B. sexangula (with knee roots), and R. stylosa (with prop roots) for study. Three 10 m × 10 m permanent plots were established in each forest. The tree height, stem diameter at breast height (DBH: ~ 1.3 m above ground) of each tree, and tree density were recorded in these permanent plots (Table 1).

Root zone expansion monitoring
Surface elevation tables (SET) were installed to measure surface elevation changes, following Lynch et al., (2015). Four polyvinyl chloride (PVC) pipes acting as shallow surface elevation legs (Shallow SET legs) were installed on surface sediment with two of the measuring directions of the custom SET arm, to monitor root zone expansion (Appendix Fig. 7). The four PVC pipes were 50 cm long were inserted in sediment with a depth of 40 cm. Then, a PVC lid was put on the top of each pipe. During measurement, the pins of SET arms can touch the lid on the tube, and the vertical distance of the root tube traversed during a time period was measured.
Since root growth can jack up the root pipes vertically, the rate of vertical movement of the roots is the rate of root zone expansion (Cahoon et al. , 2006. For the plots, twelve shallow SETs (four forests × three replicates) were installed in each permanent plot in December 2014. Initial root zone expansion data were collected through SET in May 2015 with root zone expansion data also collected after one year in June 2016. Root zone expansion was measured as the difference in the data between May 2015 and June 2016. Later, the root zone expansion data were collected twice a year from 2016 to 2020.

Root coring for root biomass samples
Root coring was used to estimate the distribution of roots. By separating the fine and coarse roots, we calculated the biomass proportion of fine and coarse roots in the 1 m deep sediment cores of the four mangrove forest types. We collected five root cores in four mangrove forests with a steel corer (diameter of 5 cm, depth of 1 m) in November 2020. The top 1 m was separated into ten 0.1 m segments. A total of 200 sediment samples were collected (4 species × 5 horizontal × 10 layers). All sediment samples were transported in polythene bags and taken back to the laboratory, where they were stored at 4 °C until further use. We obtained all the root samples from permanent plots and used tap water for gentle clearing. Next, all the root samples were classified into fine roots (diameter at < 2 mm) and coarse roots (diameter at ≥ 2 mm). Then, we separated the fine roots into live and dead fine roots using 11% and 6% solutions of colloidal silicate (Ludox® TM, Sigma-Aldrich Inc., St. Louis, MO, USA) (Robertson and Dixon 1993;Xiong et al. 2017). All root samples, including coarse roots, live fine roots, and dead fine roots, were oven dried (60 °C) until a constant weight was reached prior to measuring the biomass. Dead fine roots were ground by hand using a mortar and pestle and then analyzed for C content (%) on a Vario EL cube CHN autoanalyzer (Elementar, Langenselbold, Germany).
In-growth bags for root production We monitored root production using in-growth bags in permanent plots in June 2017 (Neill 1992). Thirty-six in-growth bags (nylon mesh bags, diameter of 5 cm, length of 40 cm) in total (4 forests × 3 plots × 3 replicates) were filled with root-free soil collected from adjacent unvegetated mudflat and vertically installed to a sediment depth of 40 cm (He et al. 2021). We retrieved each in-growth bag and collected all the samples twice a year in summer and winter from 2017 to 2020. All root samples from each in-growth bag were gently cleaned using tap water. Then, we separated coarse, live fine roots, and dead fine roots through the same methods that we applied to root samples from root coring. After collection, root samples were stored at 4 °C until further analysis. All root samples of in-growth bags, including coarse, live fine roots, and dead fine roots, were oven dried (60 °C) until a constant weight prior to measuring the biomass. We obtained the turnover rate of fine roots through root coring and in-growth bags. The fine root turnover rate (yr −1 ) was calculated through fine root biomass and fine root productivity using formula (1). (Cormier et al. 2015).

Elemental analysis of sediment samples
We collected sediment samples near every root coring sample using the same method applied for root coring. All sediment samples were transported in polythene bags and taken back to the laboratory, where they were stored at 4 °C until further use. All sediment samples were oven dried (60 °C) until constant weight, at which point the bulk density (g cm −3 ) and water content (%) were measured. After that, all the sediment samples were ground by hand using a mortar and pestle and analyzed for C content (%) on a Vario EL cube CHN autoanalyzer (Elementar, Langenselbold, Germany).

Data analysis
Differences in fine root production, root zone expansion, live fine root biomass, and fine root biomass among different sediment depths were determined by one-way ANOVA. Differences were regarded as significant when P < 0.05. All statistical tests were conducted using SPSS 20.2 (SPSS Inc. Armonk, USA).

Results
The biomass of coarse roots and fine roots Fine root biomass contributed over 60% to the root biomass at different sediment depths of the 1 m cores when compared with the coarse root biomass in the Bruguiera forest, Sonneratia plantation, and Rhizophora forest. More than 65% of roots were distributed in the upper 40 cm of sediment in the Bruguiera forest, Kandelia forest, and Sonneratia plantation.
Live and dead fine root biomass and fine root production By separating live and dead fine roots from the root coring samples, we found that there were more (1) Turnover rate = Fine root productivity∕ Fine root biomass dead fine roots than alive ones in the upper 1 m of sediment of the Bruguiera, Kandelia forests and Sonneratia plantations (Fig. 3), while the opposite pattern was found in the Rhizophora forest (Fig. 3). A majority of live fine roots were distributed at sediment depths of 20-40 cm in Kandelia and Bruguiera forests and Sonneratia plantations, while more live fine roots of the Rhizophora forest were found in the 0-20 cm depth sediment layers than in other layers. In Kandelia and Rhizophora forests and Sonneratia plantations, the biomasses of live and dead fine roots at the 20-40 cm depth were significantly higher than those at 60-100 cm depth. Importantly, the Rhizophora forest had the largest biomass of live fine roots compared with the other three forest types. No significant difference Fig. 3 Biomass of live fine roots and dead fine roots (mean ± se) of the mangrove forests in Dongzhaigang Bay. Different letters denote significant differences (P < 0.05) among the four forests was observed in sediment depth related to the distribution of live and dead fine roots in the Bruguiera forest (Fig. 3). The biomasses of live and dead fine roots were significantly different for sediments from different depths in forests of the Kandelia and Rhizophora forests and Sonneratia plantations (Fig. 3). The biomass of live fine roots was significantly related to sediment water content and bulk density ( Fig. 4A; Fig. 4B).
Fine roots were continuously produced over the study period (Fig. 5A), and no statistical differences were observed in fine root productivity among the four mangrove forests (Fig. 5B).

Root zone expansion of live fine roots
The results from shallow SETs showed that the root zone expansion rate in mangrove forests in Dongzhaigang Bay averaged 0.94 mm yr −1 , with the maximum in the forest of the Rhizophora forests (1.28 mm yr −1 ), followed by the Sonneratia plantations (0.98 mm yr −1 ), Bruguiera forests

Fig. 5
Fine root production and fine root productivity (mean ± se) of the four mangrove forests in Dongzhaigang Bay. Different letters denote significant differences (P < 0.05) among the four forests (0.94 mm yr −1 ), and Kandelia forest (0.55 mm yr −1 ) (Fig. 6B). No statistical differences were observed in root zone expansion among the four mangrove forests (Fig. 6B). Live fine root biomass of Rhizophora forests was significantly higher than other three mangrove forests (Fig. 6A). Root zone expansion was significantly related to the live fine root biomass to a sediment depth of 40 cm (R 2 = 0.6250, P = 0.0022), which contributed 62.5% of live fine root to the root zone expansion in mangrove forests in Dongzhaigang Bay (Fig. 6C).

Turnover rates of fine roots
The C concentration of dead fine roots ranged from 28.5% to 31.5% (Table 2), while the fine root turnover rates ranged from 0.10 yr −1 to 0.22 yr −1 in the four forests (Table 2). However, the C concentration and the turnover of fine roots were not significantly different (P > 0.05) among the four forests (Table 2).

Effects of mangrove fine roots on belowground C stocks
Mangroves are one of the blue carbon or oceanic ecosystems with a higher C stock than most terrestrial ecosystems, including high aboveground and belowground biomass C stock and sediment C stock. In the face of global climate change and the recent increase in atmospheric CO 2 concentrations, mangrove ecosystems deserve our attention for their ability to sequester C. Belowground roots bind the sub-surface sediment based on their relatively high productivity, which can help to maintain sediment volume and avoid sediment erosion (Krauss et al. 2003;Xiong et al. 2017). The main ingredient of mangrove peat comes from the mangroves to roots (McKee et al. 2007;McKee and Faulkner 2010). Belowground mangrove roots have efficient turnover rates, while the production of belowground roots impacts the biomass C cycle in mangrove ecosystems (Krauss et al. 2014). This is especially true in fine roots, which are vital to C gain based on the death of buried fine roots in the sub-surface sediment. In this study, we found that fine roots contributed more to belowground biomass than coarse roots in the surface sediments (1 m depth) of four mangrove forest types in Dongzhaigang Bay. Similarly, Liu et al (2017) found that fine roots contributed mostly to the annual accumulation of plant debris in the sediment at the same  locations employed in the present study. Hence, we presumed that fine roots affected mangrove sediment C more than the coarse roots in Dongzhaigang Bay, and the effects on the accumulation of organic matter in sediment here primarily depend on fine root production. This is one of the reasons for the differences in belowground C stock among various mangrove forests. Different vegetation types owned soil C stock throughout the vertical distribution of soil organic C . Vertical root distribution affected the sediment organic C accumulation (Jobbágy and Jackson 2000). In the shallow sediment layers, most live fine roots were found to be wound together, especially at sediment depths of 0-40 cm, which resulted in the expansion of the root zone. In our study, the result of Shallow-SET among four mangrove forest types suggested that the volume in the shallow soil layers expansion followed the belowground root growth. Root zone expansion is vital for mangrove forests to enable the roots to solidity and tighten sediment which tends to erode because of the damp environment and because the soil is easily influenced by regular tidal forces. Live fine roots under a sediment depth of > 40 cm played an important role in root zone expansion, even increasing the elevation in sub-surface sediment with annual fine root production (Krauss et al. 2017). In the present study, the biomass of live fine roots was positively significant relative to the rate of root zone expansion (P = 0.022) (Fig. 6C). Rhizophora forest exhibited a higher rate of root zone expansion than those of the other three forest types, which had more live fine roots than other forest types in the upper 40 cm of sediment.
Fine root production represented the major pathway of plant C input to sediment in forests generally (Leppälammi-Kujansuu et al. 2014). Fine roots are the dominant contributor to the accumulation of organic matter sediment in mangroves because fine roots have higher production and lower decomposition rates than aboveground litter in mangroves . The proportion of fine roots was quantified by distinguishing live from dead fine roots, and fine roots were found to contribute to the sediment C stock in mangrove forests after living fine roots died and were then left buried in the sediment in Dongzhaigang Bay. However, the dead fine root biomass decreased with increasing sediment depth, revealing the sediment C stock in sediment varied at different sediment depths in mangrove forests in Dongzhaigang Bay. The growth and burial of fine roots are thought to be the dominant controls of C gain in mangroves because plant litter inputs can affect sediment organic C decomposition by the priming effect . Because the priming effect mainly occurs in leaf litter, litter from belowground roots should not be neglected. In addition, fine root production is associated with the exudation of fine roots, which can influence the rate of organic matter decay in sediments (Arnaud et al. 2020). Hence, we revealed the influence of belowground biomass on the sub-surface sediment C stock by studying the distribution of belowground biomass in 1 m sediment depth.

Relationship between root zone expansion and environmental factors
The slow root growth rates were caused by low oxygen concentrations in the root zone (Mckee 1996). Low oxygen concentrations in the sediment caused by sea-level rise (SLR) caused many differences in root growth in mangroves (Day and Megonigal 1993). As we know, mangroves are sensitive to SLR (Rogers et al. 2019). When SLR occurs, the depth and duration of tidal inundation increase, which has become one of the most prominent environmental stressors facing mangroves worldwide. In addition, SLR can alter the natural distribution pattern of mangroves and especially affect the intertidal distribution series of different species (Chen and Wang 2017) by influencing fine root growth (Chen et al. 2013). Sea level rise also results in decreased root biomass and decomposition rates because SLR affected the lifespan of roots (Castaneda-Moya et al. 2013) while it increased the levels of C found in mangrove sediment (Chen et al. 2020). In addition, the elevation change is the key to the response to SLR, the growth of mangroves can result in increasing the local elevation slightly (Fu et al. 2019;Krauss et al. 2014;Lovelock et al. 2015;Middleton and McKee 2001), which is followed by the process of surface accretion; this process will result in an accumulation of organic matter in the sediment . Mangroves can increase their ground elevations adaptively with SLR through biophysical processes, including the deposition of particulate matter onto the sediment surface and belowground root accumulation (Krauss et al. 2014(Krauss et al. , 2017McKee et al. 2007).
Although most studies focus on the relationship between environmental factors and surface elevation change, few studies have focused on the surface elevation change that can result from the growth of belowground roots (Cahoon and Lynch 1997;Mckee 2011;Rybczyk and Cahoon 2002). Few studies have investigated the response of root production to nutrients, flooding, and their interactions although those studies have shown that root production is influenced by many factors (Adame et al. 2014;Cormier et al. 2015). This study is one of the rare studies that have focused on the expansion of the root zone in forests, especially on the relationship between belowground roots and root zone expansion in mangroves. Root zone growth is well known to support the volume of the sediment, and sediment sinking is often observed after the death of vegetation. For example, the sediment structure changed from compact to muddy after the death of a mangrove community in Honduras . Hence, researchers developed a shallow SET to measure the results from the expansion of the root zone (Lynch et al. 2015).
The above and belowground processes contribute as vertical accretion, shallow subsidence, or expansion leading to the mangrove elevation change (Lynch et al. 2015). The root zone swelling caused by root zone expansion generates upward forces and promoted the increment of surface elevation. Compared to the adjacent mudflat, plant distribution in mangroves influenced by the elevation change should not be neglected. Since mangroves have high belowground biomass, a shallow SET can be used to obtain the rate of root zone expansion and the contribution of belowground roots to the surface elevation change got after shallow SET measuring. This was beneficial to understanding the effects of root zone expansion on the surface elevation change. Similar to our result related to root zone expansion of mangroves in Dongzhaigang Bay, Middleton and McKee (2001) showed that the rate of root zone expansion (1-12 mm yr −1 ) followed the growth of belowground roots. Cahoon et al. (2006) and McKee et al. (2007) found that the accumulation of belowground biomass significantly influenced changes in elevation. Coldren et al. (2017) and Cherry et al. (2009) also proved the phenomenon of root zone expansion exists in coastal wetland plants. Similar to these studies, the present study found root zone expansion occurred because of belowground roots in four typical mangrove forests. In addition, this study revealed that the growth of live fine roots was positively related to root zone expansion in Dongzhaigang Bay in China (R 2 = 0.625, P = 0.0022). Thus, our results can predict the future C gain and root zone expansion of mangroves in Dongzhaigang Bay threaten by environmental factors.

Conclusions
Mangroves are vital forest ecosystems that can help to regulate global climate change. The distribution of live and dead fine roots in mangrove forests has important ecological functions related to belowground C stocks. Accurate quantification of root zone expansion and C gain is beneficial for estimating the C stock in mangrove forests. In this study, we not only provided detailed insight into the variation of biomass and production of belowground roots but also monitored the root zone expansion of four mangrove forest types in Dongzhaigang Bay. Fine roots occupied over 60% of total root biomass, obviously with more biomass than coarse roots at different sediment depths. The distribution of live fine root biomass and necromass varied with sediment depth. Fine root productivity (ranging from 1.97 to 5.30 t ha −1 yr −1 ) did not vary significantly among four mangrove forest types. In addition, we revealed the root zone expansion rates (ranging from 0.55 to 1.28 mm yr −1 ) of four typical mangrove forests and proved that live fine root biomass contributed 62.5% of root zone expansion in subsurface sediment.  Table 3 Electrical conductivity variables (mean ± se) in the surface sediment of four mangrove forests in Dongzhaigang Bay. Different letters denote significant differences (P < 0.05) among the four forests